The effect of hydrofluoric acid treatment of TiO2 grit blasted titanium implants on adherent osteoblast gene expression in vitro and in vivo

It is widely accepted that implant surface factors affect the quality of the bone-to-implant interface. Recent additional treatments superimposed on moderately rough cpTitanium surface provide further enhancement of bone-to-implant contact. The aim of this study was to compare osteoinductive and bone-specific gene expression in cells adherent to titanium dioxide-grit blasted (TiO₂) versus TiO₂ grit blasted and HF treated (TiO₂/HF) cpTitanium implant surfaces. MC3T3-E1 cells were grown in osteogenic supplements on the titanium disk surfaces for 1–14 days. Real-time PCR was used to measure RUNX-2, Osterix, and bone sialoprotein (BSP) mRNA levels. Implants were placed in rat tibia and, following harvesting at 1–7 days after placement, real-time PCR was used to measure RUNX-2, alkaline phosphatase (ALP), and BSP mRNA levels in implant adherent cells. In cell culture, RUNX-2 and Osterix levels were significantly increased (p<0.05) on the TiO₂/HF surfaces as compared to the TiO₂ and smooth surfaces through the cultural period, while BSP expression was elevated on both TiO₂ and TiO₂/HF surfaces when compared to a machined surface control. In cells adherent to implants retrieved from rat tibia, RUNX-2 mRNA levels were 2-fold and 8-fold greater on the TiO₂/HF surfaces at 1–3 and 7 days following implantation. This was paralleled by significantly greater levels of ALP at 3 and 7 days and BSP mRNA at 7 days following implantation. As a marker of osteoinduction, the increased levels of RUNX-2 in cells adherent to the TiO₂/HF surfaces suggest that the additional HF treatment of the TiO₂ grit blasted surface results in surface properties that support adherent cell osteoinduction. In vivo assessments of implant adherent cell phenotypes provide further insight into the mechanisms affecting alloplast–tissue interactions.     

DNA methylation and the regulation of gene expression

An inverse relationship between a eukaryotic gene's level of methylation and its level of expression has long been recognized, and generally believed to result from reduced binding of postulated activator proteins to methylated target DNAs. There are, however, some genes where there is no apparent correlation between levels of methylation and gene expression, and even a small class where gene activation is correlated with increased methylation of the DNA. I propose a unifying hypothesis to explain these apparently divergent cases: methylation acts to reduce or abolish binding of regulatory proteins to their DNA target sites. In the majority of genes, methylation acts to block binding of activating factors; "indifferent" genes lack such methylation sites, while the minority class, which is more active when methylated, contains methylation sites which block binding of repressor proteins.     

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation.     

基于DNA芯片的细菌基因表达谱技术的建立与评价

目的建立和优化基于全基因组DNA芯片的细菌基因表达谱技术，并用实时定量RT-PCR对此技术平台进行评价。方法提取并纯化鼠疫耶尔森菌总RNA，以六核苷酸随机引物（N6）和／或基因组特异引物（GDP）逆转录合成cDNA，用CY染料直接或间接标记后，与包括鼠疫耶尔森菌4005个ORF的全基因组芯片杂交，通过芯片扫描仪获得表达谱分析结果，归一化后进行分析。结果用实时定量PCR技术验证。结果采用N6＋GDP混合引物以间接法标记获得了较为理想的结果，表达谱技术与实时定量PER技术所得结果高度相关，证明了此技术的可靠性和实验设计与数据分析的合理性。结论合理的设计和归一化的分析方法是基于DNA芯片的细菌基因表达谱分析成功的关键，该技术的建立为细菌功能基因组研究奠定了基础。     

体内电穿孔对报告基因表达和HIV GagDNA疫苗诱导的免疫反应的影响

目的 研究体内电穿孔递送途径在小鼠模型中对于报告基因表达水平及HIV Gag DNA疫苗所诱导的免疫反应的影响。方法 构建荧光素酶（1uciferase）表达质粒p1．0-1ue，将其通过直接肌肉注射、肌注后以双针电极进行电穿孔、肌注后以钳式电极进行电穿孔等3种不同方法注射小鼠，72h后取注射部位的肌肉测定luciferase的表达情况。同时构建携带密码子优化的HIV-1 B′／C重组亚型CN54株gag基因的DNA疫苗质粒p1．0-gag，在10μg、100μg两个剂量水平上通过以上3种不同的方法免疫BALB／c雌性小鼠，ELISA方法检测Gag特异的抗体反应，ELISPOF方法和细胞内因子染色（intracellular cytokine staining，ICS）技术检测细胞免疫应答。结果 通过体内电穿孔可以使luciferase在小鼠肌肉中的表达水平显著提高，最大提高幅度达到66倍。Gag DNA疫苗免疫结果显示，电穿孔可以显著提高Gag特异的体液免疫应答，其中使用双针电极的效果要显著好于钳式电极，前者所诱导的抗体滴度比不使用电穿孔组提高可达28倍。但体内电穿孔对于Gag特异的细胞免疫应答并没有显著影响。结论 体内电穿孔（尤其是使用双针电极）可以大幅度提高报告基因在体内的表达水平和DNA疫苗诱导的抗原特异性体液免疫应答。     

应用基因芯片技术筛查人子宫内膜容受性相关基因

目的探讨人子宫内膜容受性相关基因的差异表达。方法应用含14000条基因的cDNA表达谱基因芯片分析分泌早期与分泌中期子宫内膜基因的差异表达。结果所检测的14000个基因中,分泌早期子宫内膜与分泌中期子宫内膜之间存在显著差异表达基因313个。其中,分泌中期子宫内膜表达上调基因数为175个,下调基因数为138个。结论子宫内膜容受性的建立受许多因素的调控,应用基因芯片技术可以快速、高通量的筛查出相关基因,从而有可能找到合适的分子标志物作为子宫内膜容受性的临床诊断指标。     

Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis

Summary Linear hybrid plasmids based on the killer plasmid pGKL1 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae. Like pGKL1, the hybrids are located in the cytoplasm, have terminal inverted repeats (TIR) and possess covalently linked proteins at their 5 ends. The construction of cytoplasmic hybrid plasmids is based on the use of a pGKL1 promoter to control the marker gene used for recombination. Nuclear promoters are not recognised in the cytoplasm.     

The strength of T cell stimulation determines IL-7 responsiveness, secondary expansion, and lineage commitment of primed human CD4+IL-7Rhi T cells

Mouse memory T cell precursors express IL-7 receptor-alpha (IL-7R), proliferate with homeostatic cytokines and undergo secondary expansions with antigen. Here, we analyzed how the strength of antigenic stimulation regulates IL-7R expression, cytokine responsiveness and expansion potential of DC-primed human CD4(+ )T cells. IL-7R expression on proliferating T cells was highest at intermediate strength of stimulation, and purified CCR7(+)IL-7R(hi) and CCR7(-)IL-7R(lo) subsets had characteristics of memory and effector cells, respectively. However, CCR7(+)IL-7R(hi) cells generated under different priming conditions had strikingly different properties. Thus, increasing strength of stimulation promoted IL-7 responsiveness that correlated with reduced phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression and enhanced s6 kinase activation, suggesting a tunable IL-7R coupling to PI3 kinase-dependent signaling pathways. Furthermore, functional and gene expression analysis revealed that intermediate-stimulated CCR7(+)IL-7R(hi) cells were similar to non-polarized central memory cells with high expansion potential. Conversely, high-stimulated CCR7(+)IL-7R(hi) cells shared characteristics with circulating pre-Th1 cells and differentiated spontaneously to Th1 effector cells. These results show that the strength of stimulation determines properties of activated IL-7R(hi) T cells, and suggest that memory T cell subsets could be derived from CCR7(+) precursors that received different strengths of stimulation.     

力生长因子E肽与应力刺激对成骨细胞基因表达的影响

目的应用基因芯片技术分析在力生长因子E肽(MGF-Ct24E)和应力作用下成骨细胞基因表达的差异。方法体外培养原代成骨细胞,分别对细胞施加周期性拉伸刺激(应变12%,频率0.5 Hz)和MGF-Ct24E(50 mg/L)直接作用,基因芯片技术分析力学刺激和MGF-Ct24E作用对原代成骨细胞基因表达谱的影响,并用定量PCR验证基因芯片实验结果。结果与对照组相比,力学加载组共发现差异表达基因1 866个,其中上调基因1 113个,下调基因753个;MGF-Ct24E处理组共发现差异表达基因1 178个,其中上调基因796个,下调基因382个。GO分析发现两者的差异基因表达谱具有一致性,并且差异表达基因主要涉及细胞增殖与分化调节、细胞对应力刺激的响应和力学转导通路等。定量PCR实验结果验证的差异表达基因与芯片实验结果一致。结论基因表达谱分析显示应力刺激和MGF-Ct24E作用对成骨细胞的基因表达具有类似的调控效应,为后续使用MGF-Ct24E治疗骨修复以弥补应力刺激不足的研究提供了新的思路。     

放射抑制小鼠骨髓间充质干细胞成骨潜能并导致骨损伤

目的 观察放射对小鼠骨髓间充质干细胞(BMMSCs)体外成骨潜能及体内骨组织的影响.方法 分离、培养正常的及接受4Gy放射后28d的小鼠BMMSCs,用碱性磷酸酶(ALP)和Von Kossa染色法鉴定BMMSCs体外成骨分化潜能的改变,并通过骨组织形态学和骨密度(BMD)检测放射后小鼠体内骨组织的相关变化.结果 4Gy照射28d后小鼠BMMSCs的成骨潜能明显降低,同时体内骨组织结构破坏,小鼠骨密度降低.结论 放射损伤后小鼠BMMSCs的成骨潜能显著降低,可能在干细胞水平参与了放射后骨损伤的发生.     

MAPK regulation of gene expression in airway smooth muscle

Mitogen-activated protein kinases (MAPK) are important components of signaling modules activated by neurotransmitters, cytokines, and growth factors, as well as chemical and mechanical stressors. In the airway, these external signals produce acute responses that modify smooth muscle contraction and may also induce chronic responses that modify airway structure. Both acute and chronic events in airway remodeling result from altered expression of multiple genes encoding protein mediators of cell-cell signaling, extracellular matrix remodeling, cell cycle control and intracellular signaling pathways. This review will focus on inflammatory and growth factor mediators of cell-cell signaling regulated by the ERK and p38 MAPK pathways in airway smooth muscle (ASM). These signaling mediators affect ASM tissue mechanics, cell migration, and gene expression patterns in a paracrine and autocrine fashion, although the relative importance of each MAPK pathway varies with the stimulus. These events thereby contribute to normal airway function and participate in pathological changes in ASM that accompany symptoms of asthma.     

Regulation of gene expression and cell division by Polo-like kinases

Much scientific research has focused on characterising regulatory pathways and mechanisms responsible for cell integrity, growth and division. This area of study is of direct relevance to human medicine as uncontrolled growth and division underlies many diseases, most strikingly cancer. In cancer cells, normal regulatory mechanisms for growth and division are often altered, or even fail to exist. This review summarises the mechanisms that control the genes and gene products regulating cytokinesis and cell separation in the fission yeast Schizosaccharomyces pombe, as well as highlighting conserved aspects in the budding yeast Saccharomyces cerevisiae and higher eukaryotes. Particular emphasis is put on the role of gene expression, the Polo-like kinases (Plks), and the signal transduction pathways that control these processes.     

Early gene expression events in ferrets in response to SARS coronavirus infection versus direct interferon-alpha2b stimulation

Type I interferons (IFNs) are essential to the clearance of viral diseases, however, a clear distinction between genes upregulated by direct virus-cell interactions and genes upregulated by secondary IFN production has not been made. Here, we investigated differential gene regulation in ferrets upon subcutaneous administration of IFN-α2b and during SARS-CoV infection. In vivo experiments revealed that IFN-α2b causes STAT1 phosphorylation and upregulation of abundant IFN response genes (IRGs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte transendothelial migration. During infection with SARS-CoV not only a variety of IRGs were upregulated, but also a significantly broader range of genes involved in cell migration and inflammation. This work allowed dissection of several molecular signatures present during SARS-CoV which are part of a robust IFN antiviral response. These signatures can be useful markers to evaluate the status of IFN responses during a viral infection and specific features of different viruses.     

鸭子α-干扰素基因表达及多样性分析

目的：采用分子杂交及ＰＣＲ方法，分析鸭α－干扰素基因的表达及多态性。方法：从鸭外周血分离出的单核细胞在体外经ＰＨＡ（５ｕｇ／ｍｌ）刺激不同时间后，提取总ＲＮＡ。以ＲＴ－ＰＣＲ方法检测鸭α－干扰素（ＤｕＩＦＮ－α）ｍＲＮＡ表达状况。引物根据最近公布的ＤｕＩＦＮ－α基因序列设计。从鸭外周血单核细胞中提取的基因组ＤＮＡ经限制性内切酶ＢａｍＨＩ，ＨｉｎｄⅢ，ＰｓｔＩ，ＸｂａＩ消化后，以公布的ＤｕＩＦＮ－α序列为探针，采用Ｓｏｕｔｈｅｒｎ杂交分析ＤｕＩＦＮ－α基因的多样性。结果：在未经ＰＨＡ刺激的鸭外周血单核细胞（ＰＢＭＣｓ）中，未检测到ＤｕＩＦＮ－α表达；ＰＨＡ刺激４ｈ后，即可检测到ＤｕＩＦＮ－α表达，一直持续到２４ｈ，基因组ＤＮＡ限制性内切酶多态性分析表明，ＰｓｔＩ酶切后，出现片段大小各异的杂交信号，提示ＤｕＩＦＮ－     

卵泡抑制素与乙肝表面抗原融合基因表达质粒的构建及表达

目的：构建高免疫原性的抑制素真核表达载体。方法：通过PCR扩增pCMV-S基因中的S基因片段，然后将抑制素α(1-32)片段与S的融合基因克隆到真核表达质粒peDNA13．1(-)：酶切、测序鉴定后，采用脂质体包裹法将重组质粒转染Hela细胞，用SDS-PAGE和ELISA对其表达产物进行检测。用抑制素融合表达质粒免疫大白鼠，ELISA检测血中抗抑制素抗体水平。结果：酶切鉴定和序列分析表明，融合基因的表达质粒pClS构建成功。表达质粒在HeLa细胞中获得了表达，融合蛋白的分子量约为29kD，融合蛋白具有抑制素免疫原性。重组质粒免疫使大白鼠产生了抗抑制素抗体。结论：高免疫原性的抑制素表达质粒的构建为利用抑制素基因免疫技术诱导单胎动物生多胎奠定了基础。     

Temporal Pattern of Stimulation of Osteoblast-Associated Genes During Mechanically-Induced Osteogenesis In Vivo: Early Responses of Osteocalcin and Type I Collagen

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.